Flavia Pinzari

Molecular and Microscopical Investigation of the Microflora Inhabiting a Deteriorated Italian Manuscript Dated from the Thirteenth Century

This case study shows the application of nontraditional diagnostic methods to investigate the microbial consortia inhabiting an ancient manuscript. The manuscript was suspected to be biologically deteriorated and SEM observations showed the presence of fungal spores attached to fibers, but classic culturing methods did not succeed in isolating microbial contaminants. Therefore, molecular methods, including PCR, denaturing gradient gel electrophoresis (DGGE), and clone libraries, were used as a sensitive alternative to conventional cultivation techniques. DGGE fingerprints revealed a high biodiversity of both bacteria and fungi inhabiting the manuscript. DNA sequence analysis confirmed the existence of fungi and bacteria in manuscript samples. A number of fungal clones identified on the manuscript showed similarity to fungal species inhabiting dry or saline environments, suggesting that the manuscript environment selects for osmophilic or xerophilic fungal species. Most of the bacterial sequences retrieved from the manuscript belong to phylotypes with cellulolytic activities.

Salinity and Bacterial Diversity: To What Extent Does the Concentration of Salt Affect the Bacterial Community in a Saline Soil?

In this study, the evaluation of soil characteristics was coupled with a pyrosequencing analysis of the V2-V3 16S rRNA gene region in order to investigate the bacterial community structure and diversity in the A horizon of a natural saline soil located in Sicily (Italy). The main aim of the research was to assess the organisation and diversity of microbial taxa using a spatial scale that revealed physical and chemical heterogeneity of the habitat under investigation. The results provided information on the type of distribution of different bacterial groups as a function of spatial gradients of soil salinity and pH. The analysis of bacterial 16S rRNA showed differences in bacterial composition and diversity due to a variable salt concentration in the soil. The bacterial community showed a statistically significant spatial variability. Some bacterial phyla appeared spread in the whole area, whatever the salinity gradient. It emerged therefore that a patchy saline soil can not contain just a single microbial community selected to withstand extreme osmotic phenomena, but many communities that can be variously correlated to one or more environmental parameters. Sequences have been deposited to the SRA database and can be accessed on ID Project PRJNA241061.

How Peroxisomes Affect Aflatoxin Biosynthesis in Aspergillus Flavus

In filamentous fungi, peroxisomes are crucial for the primary metabolism and play a pivotal role in the formation of some secondary metabolites. Further, peroxisomes are important site for fatty acids β-oxidation, the formation of reactive oxygen species and for their scavenging through a complex of antioxidant activities. Oxidative stress is involved in different metabolic events in all organisms and it occurs during oxidative processes within the cell, including peroxisomal β-oxidation of fatty acids. In Aspergillus flavus, an unbalance towards an hyper-oxidant status into the cell is a prerequisite for the onset of aflatoxin biosynthesis. In our preliminary results, the use of bezafibrate, inducer of both peroxisomal β-oxidation and peroxisome proliferation in mammals, significantly enhanced the expression of pex11 and foxA and stimulated aflatoxin synthesis in A. flavus. This suggests the existence of a correlation among peroxisome proliferation, fatty acids β-oxidation and aflatoxin biosynthesis. To investigate this correlation, A. flavus was transformed with a vector containing P33, a gene from Cymbidium ringspot virus able to induce peroxisome proliferation, under the control of the promoter of the Cu,Zn-sod gene of A. flavus. This transcriptional control closely relates the onset of the antioxidant response to ROS increase, with the proliferation of peroxisomes in A. flavus. The AfP33 transformant strain show an up-regulation of lipid metabolism and an higher content of both intracellular ROS and some oxylipins. The combined presence of a higher amount of substrates (fatty acids-derived), an hyper-oxidant cell environment and of hormone-like signals (oxylipins) enhances the synthesis of aflatoxins in the AfP33 strain. The results obtained demonstrated a close link between peroxisome metabolism and aflatoxin synthesis.

Unmasking the measles-like parchment discoloration: molecular and microanalytical approach

Many ancient parchments are defaced by red or purple maculae associated with localized destruction of collagen fibres. Although the main characteristics of this damage were present in most of the manuscripts analysed by many authors, no common microbial or fungal denominator has been found so far, and little or no correspondence between the microbial or fungal species isolated from materials could be addressed. In this study, culture-independent molecular methods and scanning electron microscopy (SEM) were used to identify fungal and bacterial communities on parchments affected by the purple stains. Protocols for c extraction and nucleic-acid-based strategies were selected for assays examining the community structure of fungi and bacteria on biodeteriorated parchment. Both SEM and molecular analysis detected the presence of bacterial and fungal cells in the damaged areas. Halophilic, halotolerant proteolytic bacterial species were selected by the saline environment provided by the parchment samples. As common microbial denominators, members of the Actinobacteria, mainly Saccharopolyspora spp. and species of Aspergillus, were detected in all investigated cases. It is proposed that a relationship exists between the phenomenon of purple spots on ancient parchments and that of the ‘red heat’ phenomenon, known to be present in some products manufactured with marine salt.

Genotypic and Phenotypic Versatility of Aspergillus flavus during Maize Exploitation

Aspergillus flavus is a cosmopolitan fungus able to respond to external stimuli and to shift both its trophic behaviour and the production of secondary metabolites, including that of the carcinogen aflatoxin (AF). To better understand the adaptability of this fungus, we examined genetic and phenotypic responses within the fungus when grown under four conditions that mimic different ecological niches ranging from saprophytic growth to parasitism. Global transcription changes were observed in both primary and secondary metabolism in response to these conditions, particularly in secondary metabolism where transcription of nearly half of the predicted secondary metabolite clusters changed in response to the trophic states of the fungus. The greatest transcriptional change was found between saprophytic and parasitic growth, which resulted in expression changes in over 800 genes in A. flavus. The fungus also responded to growth conditions, putatively by adaptive changes in conidia, resulting in differences in their ability to utilize carbon sources. We also examined tolerance of A. flavus to oxidative stress and found that growth and secondary metabolism were altered in a superoxide dismutase (sod) mutant and an alkyl-hydroperoxide reductase (ahp) mutant of A. flavus. Data presented in this study show a multifaceted response of A. flavus to its environment and suggest that oxidative stress and secondary metabolism are important in the ecology of this fungus, notably in its interaction with host plant and in relation to changes in its lifestyle (i.e. saprobic to pathogenic).

Amid the possible causes of a very famous foxing: molecular and microscopic insight into Leonardo da Vinci's self‐portrait

Summary Leonardo da Vincis self‐portrait is affected by foxing spots. The portrait has no fungal or bacterial infections in place, but is contaminated with airborne spores and fungal material that could play a role in its disfigurement. The knowledge of the nature of the stains is of great concern because future conservation treatments should be derived from scientific investigations. The lack of reliable scientific data, due to the non‐culturability of the microorganisms inhabiting the portrait, prompted the investigation of the drawing using non‐invasive and micro‐invasive sampling, in combination with scanning electron microscope (SEM) imaging and molecular techniques. The fungus E urotium halophilicum was found in foxing spots using SEM analyses. Oxalates of fungal origin were also documented. Both findings are consistent with the hypothesis that tonophilic fungi germinate on paper metabolizing organic acids, oligosaccharides and proteic compounds, which react chemically with the material at a low water activity, forming brown products and oxidative reactions resulting in foxing spots. Additionally, molecular techniques enabled a screening of the fungi inhabiting the portrait and showed differences when different sampling techniques were employed. Swabs samples showed a high abundance of lichenized Ascomycota, while the membrane filters showed a dominance of A cremonium sp. colonizing the drawing.

Compartmentalization of gypsum and halite associated with cyanobacteria in saline soil crusts

The interface between biological and geochemical components in the surface crust of a saline soil was investigated using X-ray diffraction, and variable pressure scanning electron microscopy in combination with energy dispersive X-ray spectrometry. Mineral compounds such as halite and gypsum were identified crystallized around filaments of cyanobacteria. A total of 92 genera were identified from the bacterial community based on 16S gene pyrosequencing analysis. The occurrence of the gypsum crystals, their shapes and compartmentalization suggested that they separated NaCl from the immediate microenvironment of the cyanobacteria, and that some cyanobacteria and communities of sulfur bacteria may had a physical control over the distinctive halite and gypsum structures produced. This suggests that cyanobacteria might directly or indirectly promote the formation of a protective envelope made of calcium and sulfur-based compounds.

Use of biochemical indexes and changes in organic matter dynamics in a Mediterranean environment: a comparison between soils under arable and set-aside managements

Abstract The aims of this study were to evaluate the influence of different agronomic practices on organic matter turnover and on microbiological activity of two soils of the Castelporziano Tenuta Reserve. The first of the two soils is arable, currently cultivated with oats, while the second is a set-aside soil which has been semi-reforested in the last ten years. Results revealed that set-aside soil was characterised by slower organic carbon turnover and better humification efficiency with respect to the arable soil. This study confirms that the restoration practice can establish a self-sustaining plant and microrganisms community, which is naturally evolving in the direction of the original, stable ecosystem.

A Combined Approach to Assess the Microbial Contamination of the Archimedes Palimpsest

A combined approach, using molecular and microscopic techniques, was used to identify the microbiota associated with the Archimedes Palimpsest, an unusual parchment manuscript. SEM analyses revealed the microbial damage to the collagen fibers and the presence of characteristic cell chains typical of filamentous bacteria and fungal spores. Molecular analysis confirmed a homogeneous bacterial community colonizing the manuscript. The phyla Proteobacteria and Actinobacteria were associated with this ancient parchment; the sequences were most related to uncultured clones detected in the human skin microbiome and in ephitelium, and to cultivated species of the genera Acinetobacter and Nocardiopsis. Nevertheless, a great variation was observed among the different sampled areas indicating fungal diversity. Blumeria spp. dominated in the healthy areas of the parchment while degraded areas showed disparate fungal communities, with dominant members of the genera Mucor and Cladosporium. In addition, the quantification of the β-actin gene by real-time PCR analyses (qPCR) revealed a higher fungal abundance on degraded areas than on the healthy ones.

Fungal bioleaching of mineral components in a twentieth-century illuminated parchment

In this work, we applied scanning electron microscopy (SEM), microanalysis and Raman spectroscopy to study the fungi inhabiting a richly illuminated parchment document and the damage induced by their activity. To that aim, we collected samples of fungal mycelium from the deteriorated areas on a removable adhesive tape specifically intended for lifting fungi without damaging the support. SEM analysis of the adhesive tape samples showed the co-occurrence of several species of fungi. One strain closely resembling Acremonium species was observed only in the tape micrographs but no agar cultures were obtained. Its fungal structures showed the production of abundant oxalates with an outstanding leaching of the calcium-based materials of parchment (typically manufactured with gypsum and lime). Needle-like crystals of calcium oxalate produced by the fungus forming a uniform and quite regular grid around conidial slimy heads were documented. As a result, the areas affected by moulds were weakened, stained and characterised by a powdery patina rich in calcium. Confocal μ-Raman confirmed the presence of oxalates while EDS showed the presence of calcium in crystals. We conclude that the defacement of the parchment was due to both collagenolytic activity, and to the biotransformation of calcium-based minerals by fungi.